The body's immune system activates a variety of mechanisms for attacking pathogens (Janeway, Jr, C A. and Travers P., eds., in Immunobiology, “The Immune System in Health and Disease,” Second Edition, Current Biology Ltd., London, Great Britain (1996)). However, not all of these mechanisms are necessarily activated after immunization. Protective immunity induced by immunization is dependent on the capacity of an immunogenic composition to elicit the appropriate immune response to resist or eliminate the pathogen. Depending on the pathogen, this may require a cell-mediated and/or humoral immune response.
Many antigens are poorly immunogenic or non-immunogenic when administered by themselves. Strong adaptive immune responses to antigens almost always require that the antigens be administered together with an adjuvant, a substance that enhances the immune response (Audbert, F. M. and Lise, L. D. 1993 Immunology Today, 14: 281-284).
The need for effective immunization procedures is particularly acute with respect to infectious organisms that cause acute infections at, or gain entrance to the body through, the gastrointestinal, pulmonary, nasopharyngeal or genitourinary surfaces. These areas are bathed in mucus, which contains immunoglobulins consisting largely of secretory immunoglobulin IgA (Hanson, L. A., 1961 Intl. Arch. Allergy Appl. Immunol., 18, 241-267; Tomasi T. B., and Zigelbaum, S., 1963 J. Clin. Invest., 42, 1552-1560; Tomasi, T. B., et al., 1965 J. Exptl. Med., 121, 101-124). This immunoglobulin is derived from large numbers of IgA-producing plasma cells, which infiltrate the lamina propria regions underlying the mucosal membranes (Brandtzaeg, P., and Baklein, K, 1976 Scand. J. Gastroenterol., 11 (Suppl. 36), 1-45; and Brandtzaeg, P., 1984 “Immune Functions of Human Nasal Mucosa and Tonsils in Health and Disease”, page 28 et seq. in Immunology of the Lung and Upper Respiratory Tract, Bienenstock, J., ed., McGraw-Hill, New York, N.Y.). The secretory immunoglobulin IgA is specifically transported to the luminal surface through the action of the secretory component (Solari, R, and Kraehenbuhl, J-P, 1985 Immunol. Today, 6, 17-20).
Parenteral immunization regimens are usually ineffective in inducing secretory IgA responses. Secretory immunity is most often achieved through the direct immunization of mucosally associated lymphoid tissues. Following their induction at one mucosal site, the precursors of IgA-producing plasma cells extravasate and disseminate to diverse mucosal tissues where final differentiation to high-rate IgA synthesis occurs (Crabbe, P. A., et al., 1969 J. Exptl. Med., 130, 723-744; Bazin, H., et al., 1970 J. Immunol., 105, 1049-1051; Craig, S. W., and Cebra, J. J., 1971 J. Exptl. Med., 134, 188-200). Extensive studies have demonstrated the feasibility of mucosal immunization to induce this common mucosal immune system (Mestecky, J., et al., 1978 J. Clin. Invest., 61, 731-737), but with rare exceptions the large doses of antigen required to achieve effective immunization have made this approach impractical for purified antigens.
Among the strategies investigated to overcome this problem is the use of mucosal adjuvants. A number of adjuvants that enhance the immune response of antigens are known in the prior art (Elson, C. O., and Ealding, W., 1984 J. Immunol., 132, 2736-2741). These adjuvants, when mixed with an antigen, render the antigen particulate, helping retain the antigen in the body for longer periods of time, thereby promoting increased macrophage uptake and enhancing immune response. However, untoward reactions elicited by many adjuvants or their ineffectiveness in inducing mucosal immunity have necessitated the development of better adjuvants for delivery of immunogenic compositions. Unfortunately, adjuvant development to date has been largely an empirical exercise (Janeway, Jr., et al, cited above at pages 12-25 to 12-35). Thus, a rational and a more direct approach is needed to develop effective adjuvants for delivery of antigenic compositions.
It has been reported that the toxin secreted by the Gram-negative bacterium Vibrio cholerae (V. cholerae), the causative agent of the gastrointestinal disease cholera, is extremely potent as an adjuvant. Cholera toxin (CT) has been reported as a 382 amino acid sequence (SEQ ID NO: 1) (Mekalanos, J. J., et al., 1983 Nature, 306, 551-557), which has an 18 amino acid signal (amino acids 1 to 18 of SEQ ID NO: 1). The cholera toxin holotoxin molecule is a hexaheteromeric complex that consists of a a peptide subunit designated CT-A (SEQ ID NO: 2 or amino acids 19 to 258 of SEQ ID NO: 1), which is responsible for the enzymatic activity of the toxin, and five identical peptide subunits, each designated CT-B (each having a 21 amino acid signal (amino acids 259 to 379 of SEQ ID NO: 1), followed by the CT-B peptide subunit (amino acids 280-382 of SEQ ID NO: 1)), which is involved in the binding of the toxin to the intestinal epithelial cells as well as other cells which contain ganglioside GM1 on their surface (Gill, D. M., 1976 Biochem., 15, 1242-1248; Cuatrecasas, P., 1973 Biochem., 12, 3558-3566). CT produced by V. cholerae has the CT-A subunit proteolytically cleaved within the single disulfide-linked loop between the cysteines at amino acid positions 187 and 199 of the mature CT-A (SEQ ID NO: 2) to produce an enzymatically active A1 polypeptide (Kassis, S., et al., 1982 J. Biol. Chem., 257, 12148-12152), and a smaller polypeptide A2, which links fragment A1 to the CT-B pentamer (Mekalanos, J. J., et al., 1979 J. Biol. Chem., 254, 5855-5861). Toxicity results when the enzymatically active fragment CT-A1, upon entry into enterocytes, ADP-ribosylates a regulatory G-protein (Gsα). This leads to constitutive activation of adenylate cyclase, increased intracellular concentration of cAMP, and secretion of fluid and electrolytes into the lumen of the small intestine, thereby causing toxicity (Gill, D. M., and Meren, R., 1978 Proc. Natl. Acad. Sci., USA, 75, 3050-3054). In vitro, ADP-ribosyl transferase activity of CT is stimulated by the presence of accessory proteins called ARFs, small GTP-binding proteins known to be involved in vesicle trafficking within the eukaryotic cell (Welsh, C. F., et al., “ADP-Ribosylation Factors: A Family of Guanine Nucleotide-Binding Proteins that Activate Cholera Toxin and Regulate Vesicular Transport”, pages 257-280 in Handbook of Natural Toxins: Bacterial Toxins and Virulence Factors in Disease Vol. 8 (Moss, J., et al., eds., Marcel Dekker, Inc., New York, N.Y. (1995)).
Co-administration of CT with an unrelated antigen has been reported to result in the induction of concurrent circulating and mucosal antibody responses to that antigen (Mekalanos, J. J., et al., 1983 Nature, 306, 551-557). To minimize the occurrence of undesirable symptoms such as diarrhea caused by wild-type CT in humans, it would be preferable to use as an adjuvant a form of the CT holotoxin that has substantially reduced toxicity. Mutants of CT have been suggested as a means for achieving a more useful adjuvant. One way to rationally design mutant cholera toxin holotoxins (CT-CRMs) with substantially reduced toxicity is to identify and alter amino acid residues in the toxin molecule that are completely conserved in the family of cholera (CT) and related heat-labile enterotoxins (LT-I, LT-IIa and LT-IIb) of E. coli. Another rational way to generate mutant CT-CRMs with substantially reduced toxicity is to alter amino acid residues in the holotoxin molecule that have been identified as being important for NAD-binding based on the structural alignment of the CT backbone with the backbone of related toxins possessing ADP-ribosyl transferase enzyme activity such as diphtheria toxin (DT) and pertussis toxin (PT) (Holmes, R. K., “Heat-labile enterotoxins (Escherichia coli)” in Guidebook to Protein Toxins and their Use in Cell Biology, Montecucco, C. and Rappnoli, R., Eds., Oxford Univ. Press, Oxford, England (1997); and Holmes, R. K. et al, “Cholera toxins and related enterotoxins of Gram-negative bacteria”, pp. 225-256 in Handbook of Natural Toxins: Bacterial Toxins and Virulence Factors in Disease, vol. 8, Moss. J., et al, Eds., Marcel Dekker, Inc., New York, N.Y. 1995).
Recently, one such rationally-designed, genetically-detoxified mutant of CT was disclosed wherein a single nonconservative amino acid substitution (glutamic acid to histidine) was introduced by altering the amino acid at position 29 in the A subunit (designated CT-CRME29H). The resulting mutant cholera holotoxin demonstrated substantially reduced enzymatic toxicity, but with superior adjuvanting and immunogenic properties (International Patent Publication No. WO 00/18434, incorporated in its entirety by reference).
Thus, there is a need to identify and/or rationally design additional mutant forms of the CT holotoxin that have substantially reduced toxicity, yet possess the same or enhanced adjuvanting properties as the wild-type CT holotoxin